Various devices and methods utilizing laminar flow and diffusion principles to detect the presence of and determine the concentration of various analytes in samples, e.g. whole blood, have been described previously. (Weigl, B. H. et al., U.S. Pat. No. 6,171,865; Weigl, B. H. and Yager, P., “Silicon-Microfabricated Diffusion-Based Optical Chemical Sensor,” Sensors and Actuators B—“Europetrode” (Conference) Apr. 2, 1996, Zurich, Switzerland; Weigl, B. H., et al. “Diffusion-Based Optical Chemical Detection in Silicon Flow Structures,” Analytical Methods and Instrumentation, μTAS 96 Special Edition, 1996; Weigl, B. H. et al. “Rapid Sequential Chemical Analysis Using Multiple Fluorescent Reporter Beads,” μTAS 96, Conference Proceedings, 1996; Weigl, B. H. et al. “Fluorescence Analyte Sensing in Whole Blood Based on Diffusion Separation in Silicon-Microfabricated Flow Structures,” SPIE Proceedings, Feb. 9-11, 1997, Fluorescence Sensing Technology III; and Brody, J. and Yager, P. “Low Reynolds Number Miro-Fluidic Devices,” Solid State Sensor and Actuator Workshop, Hilton Head, S. C. Jun. 2-6, 1996)
U.S. Pat. Nos. 5,716,852; 5,972,710; 6,171,865; and PCT Patent Application Ser. No. PCT/U.S. 97105245, each of which is hereby incorporated in its entirety by reference herein to the extent not inconsistent herewith, disclose a microfabricated device comprising a laminar flow channel, at least two inlets in fluid connection with the laminar flow channel for conducting into the flow channel an indicator stream and a sample stream, and an outlet. Smaller particles in the sample stream diffuse into the indicator stream, forming a detection area wherein measurements of a detectable property are made. These three patents and the PCT application disclose methods for determining the concentration of analytes in a sample stream.
Devices such as T-sensors (e.g. U.S. Pat. No. 6,171,865) and H-filters (e.g. U.S. Pat. Nos. 6,221,677 and 5,932,100) may be used to measure a binding reaction such as that between antibodies and antigens, or between proteins and protein markers. The microfluidic device can have two or more fluid input streams in which each input stream may contain an analyte of interest, detection molecules that interact with the analyte of interest, negative and positive controls, calibration controls, or the like. These devices are designed such that all flow within the microchannels is laminar flow and cross-stream transport is by molecular diffusion.
Microchannels in such microfluidic devices may have dimensions such that the depth of the channel is less than the width of the channel, wherein width refers to the widest of the two dimensions perpendicular to the fluid flow direction. In one fluid flow configuration typical of T-sensors, the input streams travel side-by-side in parallel laminar flow having a depth less than their width, in which case diffusion occurs in the wider, widthwise dimension of the microchannel. In another configuration typical of certain perpendicular H-sensors and other microfluidic devices (e.g. U.S. Pat. Nos. 5,932,100, 6,221,677 and 6,007,775), the input streams travel as stacked ribbons, in sheet-like flow, in which case diffusion is in the narrower, depthwise dimension of the microchannel.
Microfluidic devices of this nature utilize diffusion across the fluidic interface to measure such things as protein concentrations, antigen concentrations, and diffusion coefficients. The analysis is performed by optical measurement, e.g. measurement of fluorescence intensity, across the microchannel at a particular distance downstream of the initial junction of fluid streams (Prior Art FIG. 1).
In many such systems, measurement is taken by detectors positioned to view the interdiffusion region from a position out of any plane defined by the diffusion direction, e.g. perpendicular to the direction of diffusion. In order to measure the spatial distribution of, for example, fluorescence across the x-dimension, imaging in the y-direction of side-by-side streams is required. It would not be possible to image in the x-direction (through the two streams and the interdiffusion region) without the use of more sophisticated equipment, such as a confocal microscope.
The quality of measurement, therefore, depends in part on whether enough interdiffusion of the two streams has occurred to allow good spatial resolution of the fluorescence signal. More specifically, the particular aspects of the fluorescence curves, including localized slopes, maxima, and inflection points, need to be resolved in order to perform data analysis. The quality of measurement also depends on the presence of an interdiffusion zone that is wide enough for optical measurement.
One method of improving the spatial distribution of the diffusion pattern in the interdiffusion zone is to increase the time of operation of devices in which the streams run in parallel flow. Slowing the flow rate or lengthening the channel allows more time for cross-stream diffusion to occur. However, there are several potentially prohibitive restrictions for increasing the time interval. These include the inability to achieve a low enough flow rate with a practical pumping system, the lack of practicality of fabricating a very long microfluidic channel, and the lack of clinical utility of an assay that requires a long time interval.
The amount of interdiffusion can be enhanced by using stacked, wide, sheet-like streams in which the surface area of contact between the streams is increased relative to narrower streams flowing side-by-side (e.g. U.S. Pat. Nos. 6,136,272; 6,007,775; and 6,221,677). However, while diffusion is increased using this configuration of streams, the diffusion dimension is very small and visualization is difficult.
These effects are illustrated in Prior Art FIGS. 2A and 2B and 3A and 3B in which the diffusion of an indicator dye (e.g. bromocresol purple) is followed across a channel. FIGS. 2A and 3A indicate the amount of diffusion that has taken place in a given microchannel, while FIGS. 2B and 3B indicate the width of the resulting diffusion region. FIG. 2B shows the diffusion of 2.0 μl of dye across a 0.100 mm channel width (in the direction of diffusion) for 0.4 seconds (Diffusion coefficient=2.00 e−4mm2/s). FIG. 2B shows that an interdiffusion region, having a width of 60 μm is too narrow to be visually inspected when the microchannel is configured so that diffusion occurs in the narrow dimension (sheet-like flow) even though diffusion in this case is efficient (FIG. 2A).
If the diffusion channel is configured so that diffusion occurs in the wider dimension (FIG. 3) then the extent of diffusion is less than for sheet-like flow and the interdiffusion region is still only 60 μm (from 0.47 mm to 0.53 mm) as shown in FIG. 3B, which is again too narrow to be visually inspected.
If the length of the channel used to generate data for FIG. 3 is lengthened by a factor of 100, then the width of the diffusion zone is 600 μm but both the increased length of the diffusion channel and the large volume required to fill it (200 μl) are drawbacks and preclude using this method when only small quantities of fluids are available for the assay.